Arteriosclerotic vascular disease is a leading cause of death throughout the world. While sophisticated medical techniques such as arterial endarterectomy and percutaneous balloon dilatation are being applied more and more often to treat pathologic stenotic occurrences, quite often the most effective therapy is the surgical removal of the occluded section of the vessel. In such cases, the restoration of blood flow to ischemic tissue depends on the implantation of a vascular graft.
Although autologous vascular tissue is the most suitable material for use in such grafts, prior surgical intervention and advanced vascular disease often limit the availability of such tissue. Accordingly, it has become common in recent years to implant vascular grafts fabricated of synthetic materials. While commercially available synthetic grafts are extremely durable and may be used to successfully restore blood flow to occluded tissue, associated thrombogenic complications reduce their effectiveness. In particular, smaller diameter vascular grafts tend to become dysfunctional as they are blocked by the normal clotting mechanisms. Specifically, the synthetic surface of the graft promotes the deposition of fibrin leading to associated cellular adhesion and occlusion of the vessel. Consequently, the long term prognosis for non-coated synthetic grafts is relatively poor.
To circumvent the problems associated with non-coated synthetic vascular grafts, procedures are being developed for lining prosthetics with human endothelial cells to produce a non-thrombogenic cell surface such as exists in native human vessels. The endothelial lining of natural blood vessels is a highly complex, multifunctional cell surface which interacts with both the blood and the underlying vessel wall components to maintain physiological homeostasis. Tests with animals have shown that the deposition of a functional large vessel endothelial cell lining on the interior surface of synthetic vascular grafts decreases the formation of thrombogenic occlusions and minimizes the disruption of blood flow through the vessel. However, harvesting a sufficient number of large vessel cells from a donor is difficult at best.
Recent advances in molecular biology and tissue culture have allowed the isolation and subsequent propagation of large vessel endothelial cells. In practice, the use of cultured large vessel endothelial cells is expensive, complicated and subject to inherent limitations. One problem is that cell culture techniques are highly technical requiring trained personnel and the use of specialized equipment under laboratory conditions. Yet, even under the best of conditions, the yield of cultured large vessel endothelial cells may be low. Moreover, typical seeding procedures using cultured cells require the use of specialized media under complex conditions to assure the complete and even deposition of endothelial cells on the synthetic surface of the graft.
In addition, cultured cells are generally not derived from the patient receiving the graft and, accordingly, may precipitate a wide range of immunological complications. If the immune response of the patient is not attenuated, the transplanted endothelial cells will likely be attacked and stripped from the surface of the graft by the body's defenses. Conversely, if the patient's immune system is artificially suppressed it may lead to life-threatening, opportunistic infections.
In view of these and other complications associated with the use of large vessel endothelial cell treatments of prosthetic devices, alternative methods of reducing the inherent thrombogenicity of synthetic materials have been developed. In particular, it was quickly recognized that human microvascular endothelial cells could be effectively used to render synthetic grafts non-thrombogenic.
Microvascular endothelial cells are derived from capillaries, arterioles and venules and are present in an abundant supply in most body tissues. While endothelial cells may be isolated from tissues such as brain, lung, retina, adrenal glands, liver and muscle tissue, the use of fat tissue as a source for these cells is preferred due to its abundance, availability and because its removal should not adversely affect the patient being treated. Quite often, microvascular endothelial cells are present in concentrations of 10.sup.6 cells per gram of fat or higher, providing an ample source of materials for high density deposition procedures. Moreover, as the microvascular cells used to treat the synthetic graft are usually autologous, that is, taken from the recipient of the vascular prosthesis, immunological complications may be obviated.
Typically, microvascular endothelial cells are isolated from autologous adipose tissues such as perinephric fat, subcutaneous fat, omentum, or fat associated with the peritoneal cavity. Harvesting usually takes place under sterile conditions with the required amount of fat removed in one procedure. The collected tissue may then be washed before being transferred to a buffered digestive solution generally containing proteolytic enzymes such as collagenase, papain, trypsin, and mixtures thereof.
The adipose tissue is digested at 37.degree. C. for a selected period to disrupt the connective matrix and disperse the cellular components including microvascular endothelial cells. Following digestion, the cellular components may be separated by low speed centrifugation to provide a cell-rich pellet. The pellet may be washed and used in the deposition procedure or purified further using a continuous gradient. In either case, purified cells are diluted in buffer and subsequently incubated with the synthetic prosthesis to provide endothelialized surfaces.
Commonly, collection of the desired adipose tissue involves the use of a suction pump connected to a collection apparatus having a needle or cannula. For example, U.S. Pat. Nos. 5,035,708 and 4,834,703, incorporated herein by reference, disclose the collection of adipose tissue using a suction pump to provide the necessary vacuum. However, such collection devices and associated methods tend to employ strong, uncontrollable suction that is extremely rough on the microvascular cellular components of the collected tissue. The resulting disruption of the relatively fragile cellular membranes can substantially lower the viability of the harvested cells. This, in turn, dramatically reduces the efficiency of the deposition process. While such collection procedures may provide sufficient adipose tissue, samples collected using such techniques generally require several additional labor-intensive preparatory steps to assure an adequate concentration of relatively pure microvascular endothelial cells for eventual deposition.
Further, source tissue collected using suction pumps is often relatively dirty, contaminated with unwanted body fluids and non-adipose cellular debris. Rather than obtaining translucent, white samples as seen in relatively pure adipose tissue, samples collected using pump-generated vacuums often appear bloody, with concentrations of connective or membrane tissue dispersed within the fat.
The incorporated contaminants interfere with each step of microvascular endothelial cell isolation including the initial homogenization and preparation of the collected sample for digestion. Moreover, such contaminants directly inhibit the enzymatic activity of the proteolytic enzymes leading to incomplete digestion of the sample and a corresponding reduction in the yield of non-adipose cellular components subsequently obtained by centrifugation. Finally, those cells which are collected and pelleted contain increased level of non-endothelial components. The use of such contaminated pellets further lowers the efficiency of the cell deposition procedure and interferes with the homogeneous layering of endothelial cells on the prosthetic surface. Consequently, the patient may have to endure more extensive liposuction than would otherwise be required in order to provide a sufficient number of microvascular endothelial cells.
As the efficiency of the endothelialization process is lowered at each step along the way by contaminants, the importance of starting this procedure with a relatively clean sample is evident. That is, a small increase in the amount of contaminating materials initially collected can dramatically reduce the yield of viable microvascular endothelial cells available for deposition on the surface of the synthetic graft. In addition to increasing the amount of adipose tissue which must be initially collected, the inevitable reduction in cell viability due to contaminating materials must be compensated for by longer deposition times or additional purification steps, both of which reduce the operating efficiency of the entire procedure. This can be particularly detrimental if the cells are to be collected immediately prior to the implantation of the prosthetic device.
Accordingly, a need exists to improve the yield of viable endothelial cells recovered from adipose tissue collected from a patient preparatory to implantation of a synthetic prosthesis. That is, microvascular endothelial cells which are present in a fat specimen should be more efficiently separated from the fat cells, blood cells, connective tissue, and other materials that are present in the specimen, so that a larger number of such endothelial cells are available to be deposited onto the synthetic graft.
In addition to the actual problems associated with the collection of material, the use of a suction pump complicates the operating environment and interferes with the surgeon's ability to freely maneuver the adipose tissue collection apparatus. More particularly, the collection apparatus is usually attached to the vacuum source via thick, unwieldy hoses that severely compromise the maneuverability of the collection tip. Such pumps often do not allow the precise, real time control of the strength of the vacuum at the collection tip, making it difficult to maintain constant, even harvesting of the desired source tissue. This lack of convenience and precise control inevitably results in the aspiration of undesirable tissue, thereby increasing the contaminant level of the samples or resulting in the collection of less preferable adipose tissue containing lower levels of microvascular endothelial cells. Further, vacuum sources, especially those approved for use in medical procedures, are generally complicated instruments that are relatively expensive to maintain.
In view of the deficiencies of the related technology as outlined above, it is an object of the present invention to provide an efficient, cost effective method for the collection of adipose tissue containing microvascular endothelial cells.
It is another object of the present invention to provide a reliable convenient method for the collection of substantially pure adipose tissue containing high levels of microvascular endothelial cells with a minimum of blood cells, connective tissue and other contaminants.
It is still a further object of the present invention to provide a reliable convenient method for the rapid homogenization of adipose tissue to facilitate the subsequent separation of microvascular endothelial cells.